Isopentenyldiphosphate:dimethylallyldiphosphate isomerase: construction of a high-level heterologous expression system for the gene from Saccharomyces cerevisiae and identification of an active-site nucleophile

Biochemistry ◽  
1990 ◽  
Vol 29 (32) ◽  
pp. 7531-7538 ◽  
Author(s):  
Ian P. Street ◽  
C. Dale Poulter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heterologous Expression ◽  
Active Site ◽  
Expression System ◽  
Heterologous Expression System ◽  
High Level

High level production and purification of Gα0 using the baculovirus heterologous expression system

1990 ◽  
Vol 183 (6) ◽  
pp. 2172-2173
Author(s):  
K. Torossian ◽  
J. Labrecque ◽  
M. Caron ◽  
J. Plamondon ◽  
M. Dennis
Keyword(s):  
Heterologous Expression ◽  
Expression System ◽  
Heterologous Expression System ◽  
High Level ◽  
Level Production

scholarly journals Heterologous expression of the human ZIP4 zinc transporter in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Yuting Liu ◽  
Elizabeth M. Bafaro ◽  
Robert E. Dempski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heterologous Expression ◽  
Expression System ◽  
Protein A ◽  
Brain Dysfunction ◽  
Acrodermatitis Enteropathica ◽  
Wild Type ◽  
Membrane Domain ◽  
Heterologous Expression System ◽  
Gfp Fusion Protein

The human (h) transporter, hZIP4 is the primary zinc importer in the intestine and is also expressed in a variety of organs such as the pancreas and brain. Dysfunction of hZIP4 can result in the zinc deficiency disease acrodermatitis enteropathica (AE), which disrupts digestive and immune system homeostasis. Structure-function studies of hZIP4 have been greatly hindered by the absence of a robust heterologous expression system. Here, we report the heterologous expression of hZIP4 in Saccharomyces cerevisiae. Both a wild type and a mutant S. cerevisiae strain, in which the endogenous zinc transporters are deleted, were used to test the expression and localization of an hZIP4-GFP fusion protein. A full-length hZIP4-GFP and a truncated membrane domain only (mhZIP4-GFP) protein were successfully produced and targeted to the plasma membrane in yeast.

scholarly journals Characterization of Leader Processing Shows That Partially Processed Mersacidin Is Activated by AprE After Export

2021 ◽  
Vol 12 ◽  
Author(s):  
Jakob H. Viel ◽  
Amanda Y. van Tilburg ◽  
Oscar P. Kuipers
Keyword(s):  
Antimicrobial Activity ◽  
Synthetic Biology ◽  
Heterologous Expression ◽  
Expression System ◽  
Heterologous Expression System ◽  
Proteolytic Activation ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Precursor Peptide ◽  
Potential Applications

The ribosomally synthesized and post-translationally modified peptide mersacidin is a class II lanthipeptide with good activity against Gram-positive bacteria. The intramolecular lanthionine rings, that give mersacidin its stability and antimicrobial activity, are specific structures with potential applications in synthetic biology. To add the mersacidin modification enzymes to the synthetic biology toolbox, a heterologous expression system for mersacidin in Escherichia coli has recently been developed. While this system was able to produce fully modified mersacidin precursor peptide that could be activated by Bacillus amyloliquefaciens supernatant and showed that mersacidin was activated in an additional proteolytic step after transportation out of the cell, it lacked a mechanism for clean and straightforward leader processing. Here, the protease responsible for activating mersacidin was identified and heterologously produced in E. coli, improving the previously reported heterologous expression system. By screening multiple proteases, the stringency of proteolytic activity directly next to a very small lanthionine ring is demonstrated, and the full two-step proteolytic activation of mersacidin was elucidated. Additionally, the effect of partial leader processing on diffusion and antimicrobial activity is assessed, shedding light on the function of two-step leader processing.

scholarly journals TheNitrosopumilus maritimusCdvB, but Not FtsZ, Assembles into Polymers

Archaea ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Kian-Hong Ng ◽  
Vinayaka Srinivas ◽  
Ramanujam Srinivasan ◽  
Mohan Balasubramanian
Keyword(s):  
Cell Division ◽  
Heterologous Expression ◽  
Fission Yeast ◽  
Mammalian Cells ◽  
Expression System ◽  
Related Protein ◽  
Heterologous Expression System

Euryarchaeota and Crenarchaeota are two major phyla of archaea which use distinct molecular apparatuses for cell division. Euryarchaea make use of the tubulin-related protein FtsZ, while Crenarchaea, which appear to lack functional FtsZ, employ the Cdv (cell division) components to divide. Ammonia oxidizing archaeon (AOA)Nitrosopumilus maritimusbelongs to another archaeal phylum, the Thaumarchaeota, which has both FtsZ and Cdv genes in the genome. Here, we used a heterologous expression system to characterize FtsZ and Cdv proteins fromN. maritimusby investigating the ability of these proteins to form polymers. We show that one of the Cdv proteins inN. maritimus, the CdvB (Nmar_0816), is capable of forming stable polymers when expressed in fission yeast. TheN. maritimusCdvB is also capable of assembling into filaments in mammalian cells. However,N. maritimusFtsZ does not assemble into polymers in our system. The ability of CdvB, but not FtsZ, to polymerize is consistent with a recent finding showing that several Cdv proteins, but not FtsZ, localize to the mid-cell site in the dividingN. maritimus. Thus, we propose that it is Cdv proteins, rather than FtsZ, that function as the cell division apparatus inN. maritimus.

scholarly journals Heterologous Expression and Purification of ? Galactosidase Protein Using Affinity Chromatography

2013 ◽  
Vol 5 (3) ◽  
pp. 499-513
Author(s):  
M. Z. Alam ◽  
L. Ragionieri ◽  
M. A. S. Santos ◽  
A. Iqbal
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heterologous Expression ◽  
Affinity Chromatography ◽  
Recombinant Dna ◽  
Expression System ◽  
Eukaryotic Expression ◽  
Lacz Gene ◽  
Recombinant Dna Technology ◽  
E Coli ◽  
Elution Buffer

Enzymes and other protein purification using recombinant DNA technology have become popular due to scarcity of natural protein. Saccharomyces cerevisiae is a demanding host, since it facilitates protein expression by its relative simplicity, safe organisms, inexpensive and has many properties of eukaryotic expression system. As an alternative host we express E. coli lacZ gene with GST tag in Saccharomyces cerevisiae and successfully purified from soluble extracts. The concentration of soluble GST-? galactosidase protein was approximately 0.57 mg/ml of elution buffer yielded from 50 ml yeast cell culture. The ?-galactosidase protein from insoluble extract was low due to the increasing solubility of GST tag. Keywords: ?-galactosidase; Heterologous expression; GST tag; Affinity chromatography. © 2013 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. doi: http://dx.doi.org/10.3329/jsr.v5i3.13820 J. Sci. Res. 5 (3), 499-513 (2013)  

scholarly journals Bombyx prothoracicostatic peptides activate the sex peptide receptor to regulate ecdysteroid biosynthesis

2010 ◽  
Vol 107 (5) ◽  
pp. 2060-2065 ◽  
Author(s):  
Naoki Yamanaka ◽  
Yue-Jin Hua ◽  
Ladislav Roller ◽  
Ivana Spalovská-Valachová ◽  
Akira Mizoguchi ◽  
...  
Keyword(s):  
Expression System ◽  
Neurosecretory Cells ◽  
Neuroendocrine System ◽  
Prothoracic Gland ◽  
Heterologous Expression System ◽  
Peptide Receptor ◽  
Sex Peptide ◽  
Ecdysteroid Biosynthesis ◽  
High Level ◽  
G Protein Coupled

Insect molting and metamorphosis are induced by steroid hormones named ecdysteroids, whose production is regulated by various neuropeptides. We cloned the gene and analyzed the expression of the prothoracicostatic peptide, a unique neuropeptide shown to suppress the production of ecdysteroids in the prothoracic gland of the silkworm, Bombyx mori. We also characterized a Bombyx G protein-coupled receptor, which has previously been identified as an ortholog of the Drosophila sex peptide receptor, as a functional prothoracicostatic peptide receptor. This receptor responded specifically to the prothoracicostatic peptides when examined using a heterologous expression system. The receptor was highly expressed in the prothoracic gland on the day before each larval and pupal ecdysis, when prothoracicostatic peptides are synthesized at a high level in the epiproctodeal glands. These results suggest that the sex peptide receptor functions as a prothoracicostatic peptide receptor in Bombyx and that the peripheral neurosecretory cells as well as the central neuroendocrine system play stage-specific roles in regulating ecdysteroidogenesis.

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